Severe traumatic injuries and invasive surgical procedures such as tumor resection often create peripheral nerve gaps, accounting for 200,000 injuries in the US annually. The clinical "gold standard" for bridging peripheral nerve gaps is autografts (typically using sensory sural nerve), where 40~50% of patients regain useful function. However, the use and effectiveness of autografts is limited by several issues, including their limited availability, collateral damage at the donor site, and the presence of inhibitory chondroitin sulfate proteoglycans within the grafts. Therefore, it is critical to develop alternative approaches that match or exceed the performance of autografts. However, the leading bioengineering strategy of using nerve guidance channels has limited efficacy and has not been successful in bridging gaps longer than 10mm in rat models. In the previous grant period, we proposed to design 3D hydrogel based scaffolds with either isotropically or anisotropically distributed ECM (laminin-1) or nerve growth factor (NGF) to bridge gaps longer than 15mm in rats. We successfully designed and fabricated isotropic and anisotropic 3D hydrogels, demonstrated that indeed growth cones extend processes significantly faster in immobilized ECM gradients in vitro and that anisotropic scaffolds with gradients of laminin-1 and NGF are able to bridge 17-20mm gaps, whereas isotropic scaffolds with uniformly distributed LN-1 and NGF do NOT. Though, unfortunately, anisotropic hydrogel scaffolds only meet success 44% of the time in bridging 17-20mm gaps in rats. Therefore, we have developed a new approach to anisotropic scaffolds - using aligned electrospun polymeric nanofibers (diameter 200- 600nm). In our preliminary data, we demonstrate that this novel oriented nanofiber-based 3D scaffold enhances peripheral nerve regeneration across long nerve gaps (17mm rat model) and matches the performance of autografts by anatomical and histological measures. A critical finding of this study was that oriented nanofibers enabled efficient Schwann cell migration into the scaffolds, which was a precursor to realizing the endogenous regenerative potential of severed peripheral nerves. The central hypothesis of this proposal is that functionalizing the polymeric nanofiber based scaffolds with factors that enhance Schwann cell migration and tropic/trophic functions will enable nanofiber based scaffolds to exceed the performance of autografts. We propose testing this hypothesis in a challenging 17mm nerve gap in rodents. In this next generation design, the oriented scaffolds will be biodegradable and `functionalized'to include biochemical cues, such as growth stimulatory extracellular matrix (laminin-1) and trophic protein (Neurotrophin 3, NT-3). We suggest that by concentrating the pro- regenerative cues (structural and biochemical) we can design engineered scaffolds that out-perform autografts in rigorous, clinically relevant animal models of peripheral nerve injury. When complete, this research will represent a significant step in the direction of providing alternatives to autografts for peripheral nerve repair. PUBLIC HEALTH RELEVANCE: 200,000 peripheral nerve injuries occur every year in the US alone. This research will advance our understanding of the mechanisms of peripheral nerve regeneration, and develops technologies that are likely to improve clinical outcomes after peripheral nerve injury.